The present invention is directed to a method and automated apparatus for performing isoelectric focusing of macromolecules, and particularly proteins. More particularly, the present invention is directed to an automated apparatus for supplying protein samples from a sample well to a gel tube for the first dimension isoelectric focusing of the protein sample.
Isoelectric focusing (IEF) is an electrophoretic technique for the analysis, separation and purification of various biological materials. Since many of the complex molecules of biological interest are amphoteric in nature, they are typically amenable to IEF separation.
Isoelectric separation is a known process that has been used for many years. An isoelectric focusing gel, such as an acrylamide gel, is placed or polymerized in a tube having open ends. Each open end is positioned in a bath containing a buffer solution. One buffer solution is typically a sodium hydroxide solution to contact one end of the gel tube. The other buffer solution is typically a phosphoric acid solution at the opposite end of the tube to produce a pH gradient between the two ends of the tube. When current is applied, the two buffer solutions, together with ampholytes incorporated into the gel composition or titratable gel monomers incorporated into the gel, provide an electric potential through the gel along the length of the tube. The sample to be analyzed is applied to a top end of the gel in a tube and an electric current is applied to an electrode in each of the buffer solutions. The molecules in the sample migrate through the gel under the influence of the electric potential until they reach their isoelectric point.
The separation of macromolecules, and particularly proteins, often is carried out by a two-dimensional electrophoresis separation process. The two-dimensional electrophoresis separation typically involves the sequential separation by isoelectric focusing of a sample in a gel tube followed by slab gel electrophoresis. The isoelectric focusing process is often referred to as first dimension separation. Slab gel electrophoresis, often referred to as second dimension separation, utilizes an electrophoresis gel molded between two glass plates. A gel strip or cylinder in which the protein sample has been resolved by the first dimension isoelectric focusing is placed along one edge of the slab gel. The opposite ends of the gel slab are immersed in a buffer solution and an electric current is applied between the ends to provide an electric potential through the gel slab. The proteins are then allowed to migrate through the gel slab under an applied voltage.
Charged detergents, such as sodium dodecyl sulfate, contained in the slab gel bind to the protein molecules. The detergents tend to unfold the protein molecules into rods having a length proportional to the length of the polypeptide chain and thus proportional to the molecular weight of the polypeptide. A protein complexed with a charged detergent is highly charged, which causes the protein-detergent complex to move in an applied electric field. When the slab gel, such as a polyacrylamide gel, functions as a sieve, the movement of the longer and higher molecular weight molecules is retarded compared to the shorter, lower molecular weight molecules.
Electrophoresis separation is generally labor intensive since numerous samples are run simultaneously. In the first dimension separation, the gel tubes are prepared and placed in a suitable tank of buffer solutions. The protein samples are then manually placed on the end of a gel tube. When hundreds of protein samples are prepared daily for isoelectric focusing, the manual steps significantly increase the time requirements for performing the first dimension separation. Accordingly, there is a need in the industry for improved methods and devices for conducting first dimension isoelectric focusing.
The present invention is directed to a method and apparatus for the electrophoresis separation of macromolecules and particularly proteins. More particularly, the invention is directed to an automated apparatus for first dimensional isoelectric focusing of proteins and other macromolecules.
Accordingly, a primary aspect of the invention is to provide an automated apparatus for handling and manipulating a large number of samples for electrophoresis separation.
Another aspect of the invention is to provide an automated apparatus for sequentially transferring a large number of biological samples from a respective sample container to a respective gel tube for performing electrophoresis separation of the sample.
A further aspect of the invention is to provide an automated apparatus for transferring a biological sample from a sample container to a gel tube where information identifying the sample and the location of the sample is stored in a computer.
Another aspect of the invention is to provide an automated apparatus for electrophoresis separation including a sample container magazine having a holding device for holding a sample container stationary while a sample is being removed.
A further aspect of the invention is to provide an automated apparatus for electrophoresis separation including a computer controlled arm having a pipette for piercing a septum in a sample container and removing a selected quantity of a sample from the container.
Still another aspect of the invention is to provide an automated apparatus for electrophoresis separation including a computer controlled arm having a pipette, and a sample container holding device for holding the sample container stationary while the pipette penetrates and is withdrawn from a septum in the sample container.
Another aspect of the invention is to provide an automated apparatus for transferring a plurality of biological samples to a respective gel tube where the assembly has a computer for recording and tracking the location of the samples.
A further aspect of the invention is to provide an automated apparatus for transferring a plurality of samples to a respective gel tube, wherein the apparatus includes a support member, a movable arm coupled to the support member and is movable along a longitudinal dimension of the support member, and a pipette mounted on the movable arm that is movable vertically for withdrawing a sample from a container and for dispensing a sample to a gel tube.
Another aspect of the invention is to provide an automated apparatus for electrophoresis separation having a robotic arm with a pipette that is movable in three dimensions and where the pipette is movable from a sample withdrawing position to a sample dispensing position.
A further aspect of the invention is to provide an automated apparatus for electrophoresis separation of macromolecules, where the apparatus has a plurality of electrophoresis gel tanks, each supporting a parallel row of gel tubes. The apparatus has a movable robotic arm that is able to transfer a sample from a sample vessel to a selected gel tube.
Another aspect of the invention is to provide a rack for supporting a plurality of gel tubes in an electrophoresis tank and where the rack has an open well containing a buffer solution for electrophoresis separation and a guide for guiding a pipette to an end of a gel tube that is positioned in the bottom of the well.
Still another aspect of the invention is to provide an automated transferring device for transferring samples from a sample container to a gel tube where the device includes a stationary cover member positioned above an electrophoresis tank and where the cover member includes a plurality of apertures aligned with the gel tubes.
A further aspect of the invention is to provide an automated transferring device for transferring samples from a container to an electrophoresis device where the transferring device includes a cover member having a plurality of apertures aligned in spaced apart rows and aligned with the electrophoresis device.
Another aspect of the invention is to provide an electrophoresis separation apparatus having a computer for controlling an electric power supply to the gel tanks and for the acquisition of run data for quality control.
The foregoing aspects and advantages of the invention are basically attained by providing an automated first dimensional electrophoresis separation apparatus comprising an electrophoresis assembly supporting a plurality of gel tubes containing an electrophoretic gel. Each of the tubes has a first open end and second open end and a supply magazine for containing a plurality of sample containers. Each sample container contains a sample to be subjected to electrophoresis. A transferring device is provided for sequentially removing a sample from a preselected sample container and transferring the sample to a first end of a respective gel tube. The transferring device includes a pipette that is movable in three dimensions between the supply magazine and a gel tube of the electrophoresis assembly. A microprocessor is operatively connected to the transferring device to automatically control the transfer of the sample to the respective gel tubes.
The aspects of the invention are further attained by providing an automated first dimension electrophoresis separation assembly comprising an electrophoresis assembly including at least one tank and a plurality of gel tubes vertically supported in the tank and arranged in a row. The gel tubes have an open top end. A supply magazine is provided for containing a plurality of sample containers. Each of the sample containers contains a liquid sample. A movable arm is movable in a substantially linear horizontal first direction between the supply magazine and the electrophoresis assembly. A movable pipette is coupled to the arm and is movable along a longitudinal dimension of the movable arm in a horizontal second direction substantially perpendicular to the first direction. The pipette is further movable in a vertical direction with respect to the movable arm. The pipette is movable from a first position for removing a sample from a sample container to a second position for dispensing a sample in a respective gel tube.
The aspects of the invention are still further attained by providing an apparatus for loading a biological sample in to an electrophoresis device. The apparatus comprises a base, a vertical support, and a stationary cover member spaced from the base. The cover has a top surface, a bottom surface and a plurality of apertures extending between the top and bottom surfaces and arranged in a plurality of spaced apart rows. The bottom surface of the cover member is positioned to receive a plurality of electrophoresis devices. The apparatus also includes a supply magazine for containing a plurality of sample containers that contain a biological sample. A robotic arm is movable between the supply magazine and a selected aperture of the cover member. The robotic arm has a pipette for withdrawing a sample from a sample container and delivering the sample through the aperture in the cover member to the electrophoresis device below the cover member. A microprocessor is operatively connected to the robotic arm for operating the robotic arm and the pipette.
The aspects, advantages and salient features of the invention will become apparent to one skilled in the art in view of the following detailed description of the invention in conjunction with the annexed drawings which form a part of this original disclosure.